The work described here was funded by Fina Biosolutions LLC.

<ol>
	<li>Ellis, R. W., Granoff, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development and clinical uses of Haemophilus B conjugate vaccines. , (1994).</li><li>Goebel, W. F., Avery, O. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemo-immunological+studies+on+conjugated+carbohydrate-proteins.">Chemo-immunological studies on conjugated carbohydrate-proteins.</a> Journal of Experimental Medicine. 50 (4), 533-550 (1929).</li><li>Mond, J. J., Vos, Q., Lees, A., Snapper, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+independent+antigens.">T cell independent antigens.</a> Current Opinion in Immunology. 7 (3), 349-354 (1995).</li><li>Cruse, J. M., Lewis, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Conjugate Vaccines. 10, (1989).</li><li>Lees, A., Nelson, B. L., Mond, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+soluble+polysaccharides+with+1-cyano-4-dimethylaminopyridinium+tetrafluoroborate+for+use+in+protein-polysaccharide+conjugate+vaccines+and+immunological+reagents.">Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate for use in protein-polysaccharide conjugate vaccines and immunological reagents.</a> Vaccine. 14 (3), 190-198 (1996).</li><li>Shafer, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+soluble+polysaccharides+with+1-cyano-4-dimethylaminopyridinium+tetrafluoroborate+(CDAP)+for+use+in+protein-polysaccharide+conjugate+vaccines+and+immunological+reagents.+II.+Selective+crosslinking+of+proteins+to+CDAP-activated+polysaccharides.">Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) for use in protein-polysaccharide conjugate vaccines and immunological reagents. II. Selective crosslinking of proteins to CDAP-activated polysaccharides.</a> Vaccine. 18 (13), 1273-1281 (2000).</li><li>Schneerson, R., Barrera, O., Sutton, A., Robbins, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+characterization,+and+immunogenicity+of+Haemophilus+influenzae+type+b+polysaccharide-protein+conjugates.">Preparation, characterization, and immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugates.</a> Journal of Experimental Medicine. 152 (2), 361-376 (1980).</li><li>Lees, A., Barr, J. F., Gebretnsae, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+soluble+polysaccharides+with+1-cyano-+4-dimethylaminopyridine+tetrafluoroborate+(CDAP)+for+use+in+protein-polysaccharide+conjugate+vaccines+and+immunological+reagents.+III+Optimization+of+CDAP+activation.">Activation of soluble polysaccharides with 1-cyano- 4-dimethylaminopyridine tetrafluoroborate (CDAP) for use in protein-polysaccharide conjugate vaccines and immunological reagents. III Optimization of CDAP activation.</a> Vaccines. 8 (4), 777 (2020).</li><li>Qi, X. -. Y., Keyhani, N. O., Lee, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+determination+of+hydrazine,+hydrazides,+and+their+mixtures+with+trinitrobenzenesulfonic+acid.">Spectrophotometric determination of hydrazine, hydrazides, and their mixtures with trinitrobenzenesulfonic acid.</a> Analytical biochemistry. 175 (1), 139-144 (1988).</li><li>Monsigny, M., Petit, C., Roche, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+determination+of+neutral+sugars+by+a+resorcinol+sulfuric+acid+micromethod.">Colorimetric determination of neutral sugars by a resorcinol sulfuric acid micromethod.</a> Analytical biochemistry. 175 (2), 525-530 (1988).</li><li>Hermanson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioconjugate Techniques. 3rd ed. , (2013).</li></ol>

Andrew Lees is founder and owner of Fina Biosolutions. He holds several patents relating to CDAP chemistry and benefits from licensing of the chemistry and CDAP conjugation know-how.

CDAP is a convenient reagent to derivatize and conjugate polysaccharides. This article describes the general method to use CDAP to derivatize polysaccharides with hydrazides (PS-ADH) and incorporates recently published improvements8. First, the technique emphasizes the importance of maintaining the target pH to control the activation process. We found that while many common buffers interfere with the CDAP activation reaction, DMAP could successfully be used as the buffer to manage the pH8. Furthermore, DMAP is already a reaction byproduct of CDAP activation. Finally, buffering the polysaccharide solution with DMAP before adding the CDAP facilitates precise targeting and maintenance of the reaction pH. As we describe, it is useful to adjust the pH of the concentrated DMAP stock solution such that when diluted, it reaches the targeted pH. Secondly, performing the process in the cold slowed the reaction time, making the activation process less frenetic and more forgiving. Lower temperature decreased the rate of CDAP hydrolysis, and the optimal activation time at pH 9 increases from ~3 min to ~15 min. In addition, less CDAP is required to achieve the same level of activation than when performed at room temperature. 
ADH-derivatized polysaccharides can be conjugated to proteins using carbodiimides (e.g., EDAC)7. For example, several licensed Haemophilus influenzae b (Hib) vaccines use the polyribosylribitolphosphate (PRP) derivatized with ADH to conjugate to tetanus toxoid using EDAC. CNBr was initially employed, but CDAP is a much easier reagent to use for this purpose. In our experience, a good target range for ADH derivatization is 10-30 hydrazides per 100 kDa polysaccharide or ~1-3% ADH by weight. 
The same process can be used to derivatize polysaccharides with primary amines by substituting the ADH for a diamine. It is recommended to use hexane diamine to derivatize polysaccharides with amines8. The aminated polysaccharide (PS-NH2) can be conjugated using reagents developed for protein conjugation11. Typically, the PS-NH2 is derivatized with a maleimide (e.g., succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) or N-&#947;-maleimidobutyryl-oxysuccinimide ester (GMBS)), and the protein is thiolated (e.g., with succinimidyl 3-(2-pyridyldithio)propionate (SPDP)). Thiol-maleimide chemistry is very efficient.
Proteins can also be directly coupled to CDAP-activated polysaccharides via the &#603;-amine on lysines. While the activation protocol used is generally similar to the one described here, it is necessary to optimize the level of activation, polysaccharide and protein concentration, as well as the protein:polysaccharide ratio5,6,8. 
Dextran is one of the easiest polysaccharides to activate with CDAP due to its relatively high density of hydroxyl groups, but some polysaccharides, such as Vi antigen, can be challenging. Consequently, there is no single "best" protocol for CDAP conjugation directly to proteins. We suggest first developing a protocol to achieve suitable levels of activation, as determined by the extent of hydrazide derivatization, and then proceeding to direct protein conjugation to CDAP-activated polysaccharide.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62597/activation-conjugation-soluble-polysaccharides-using-1-cyano-4">Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP)</a>. A figure&#160;was updated.
Figure 4 was updated from:
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62597/62597fig04.jpg" /> 
Figure 4: Representative results for CDAP activation of dextran. Typical standard curves for the (A) resorcinol/sulfuric acid and (B) TNBS assays. The assay results for dextran activated with 0.25 and 0.5 mg CDAP/mg dextran are shown. Glucose was used as the standard for the resorcinol assay. Dextran, in mg/mL, is divided by 100 kDa to give a molar concentration. The hydrazide concentration is determined using ADH as the standard and the results expressed as &#181;M Hz.&#160;(C)&#160;Calculation of hydrazide: dextran ratios.The level of derivatization was calculated as hydrazides per 100 kDa of dextran to facilitate the comparison between polymers of different average molecular weights. The % weight ratio of g ADH/g dextran was calculated using a MW of 174 g/mole for ADH. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62597/62597fig4v4.jpg" /> 
Figure 4: Representative results for CDAP activation of dextran. Typical standard curves for the (A) resorcinol/sulfuric acid and (B) TNBS assays. The assay results for dextran activated with 0.25 and 0.5 mg CDAP/mg dextran are shown. Glucose was used as the standard for the resorcinol assay. Dextran, in mg/mL, is divided by 100 kDa to give a molar concentration. The hydrazide concentration is determined using ADH as the standard and the results expressed as &#181;M Hz.&#160;(C)&#160;Calculation of hydrazide: dextran ratios.The level of derivatization was calculated as hydrazides per 100 kDa of dextran to facilitate the comparison between polymers of different average molecular weights. The % weight ratio of g ADH/g dextran was calculated using a MW of 174 g/mole for ADH. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig4v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62597/activation-conjugation-soluble-polysaccharides-using-1-cyano-4">Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP)</a>. A figure&#160;was updated.

Erratum: Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP)

Conjugate vaccines, such as those consisting of polysaccharides covalently linked to a carrier protein, are among the remarkable advances in vaccinology1,2. Polysaccharides, as T-cell-independent antigens, are poorly immunogenic in infants and do not induce memory, class switching, or affinity maturation of antibodies3. These shortcomings are overcome in polysaccharide conjugate vaccines4. As most polysaccharides do not have a convenient chemical handle for conjugation, they must first be made reactive or &#34;activated.&#34; The activated polysaccharide is then linked either directly with the protein (or modified protein) or is functionalized for additional derivatization before conjugation4. Most licensed polysaccharide conjugate vaccines use either reductive amination or cyanylation to activate polysaccharide hydroxyls. Cyanogen bromide (CNBr), a reagent that had previously been used to activate chromatography resins, was initially used for polysaccharide derivatization. However, CNBr requires high pH, typically ~ pH 10.5 or greater, to partially deprotonate polysaccharide hydroxyls so that they are sufficiently nucleophilic to attack the cyano group. The high pH can be detrimental to base-labile polysaccharides, and neither CNBr nor the active cyano-ester initially formed is sufficiently stable at such high pH.
CDAP (1-cyano-4-dimethylaminopyridine tetrafluoroborate; Figure 1) was introduced by Lees et al. for use as a cyanylating agent for the activation of polysaccharides5,6. CDAP, which is crystalline and easy to handle, was found to activate polysaccharides at a lower pH than CNBr and with fewer side reactions. Unlike CNBr, CDAP-activated polysaccharides can be directly conjugated to proteins, simplifying the synthesis process. CDAP-activated polysaccharides can be functionalized with a diamine (e.g., hexane diamine) or a dihydrazide (e.g., adipic dihydrazide, ADH) to make amino- or hydrazide-derivatized polysaccharides. A high concentration of the homobifunctional reagent is used to suppress crosslinking of polysaccharides. Amino polysaccharides can then be conjugated using any of the myriad techniques used for protein conjugation. Hydrazide-derivatized polysaccharides are often coupled to proteins using a carbodiimide reagent (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC))7. Further optimization of CDAP polysaccharide activation has been described by Lees et al.8 and is incorporated into the protocol described here.
CDAP conjugation overview 
The CDAP protocol can be conceptualized as two phases: (1) the activation of the polysaccharide and (2) conjugation of the activated polysaccharide with a protein or ligand (Figure 2). The goal of the first step is to efficiently activate the polysaccharide, while the goal of the second is to efficiently conjugate to the activated polysaccharide. The activated polysaccharide ties the two steps together. This conceptualization helps focus on the critical elements of each step. Figure 2 expands on this conceptualization, showing the desired activation and coupling reactions, along with the hydrolysis reactions and side reactions.
During the activation phase, the three major concerns are CDAP stability, CDAP reaction with the polysaccharide hydroxyls, and the stability of the activated polysaccharide (Figure 3). CDAP hydrolysis increases with pH, as does the hydrolysis of the activated polysaccharide and the side reactions. However, the CDAP reaction with the polysaccharide is facilitated by increasing the pH. Efficiently activating polysaccharides with CDAP requires a balance between 1) the reactivity of the polysaccharide and CDAP and 2) the hydrolysis and side reactions of both the reagent and the activated polysaccharide.
In the original CDAP activation protocol described by Lees et al.5, CDAP activation of polysaccharides was carried out at room temperature in unbuffered pH 9 solution. The rate of activation was found to be rapid under this condition, and the activation would be complete within 3 min. The reaction was also accompanied by rapid hydrolysis of CDAP, causing a rapid pH drop of the unbuffered reaction solution. It was challenging to quickly raise and to maintain the reaction pH at the target value in such a short time frame. In the described protocol, activation was performed by adding CDAP from a 100 mg/mL stock solution to the unbuffered polysaccharide solution. The pH was raised 30 s later with &#34;an equal volume of 0.2 M triethylamine&#34;. Protein to be conjugated was then added after 2.5 min to the activation reaction. Notably, the pH of the activation step was not well controlled and most likely initially exceeded the target pH. The fast reaction requiring prompt pH adjustment made the activation process difficult to control and challenging to scale up.
In contrast to the original protocol, the modified protocol described here has two major improvements. First, the pH of the polysaccharide solution is pre-adjusted to target activation pH, using DMAP as the buffer, before the addition of CDAP. DMAP has a pKa of 9.5 and thus has good buffering power around pH 9, and unlike many other buffers, DMAP was not found to promote CDAP hydrolysis8. Furthermore, DMAP is already a process intermediate and therefore does not add a new component to the reaction mixture. Pre-adjusting the pH before adding CDAP eliminates the large pH swing at the beginning of the reaction and allows for more efficient maintenance of the target pH during the reaction. The second improvement is to perform the activation reaction at 0 &#176;C, where the rate of CDAP hydrolysis is markedly slower than that at room temperature. With the longer reagent half-life at 0 &#176;C, the activation time is increased from 3 min to 15 min to compensate for the slower activation rate at the lower temperature. The longer reaction time, in turn, makes it easier to maintain the reaction pH. The use of 0 &#176;C also slows the degradation of pH-sensitive polysaccharides, making it possible to prepare conjugates of this type of polysaccharide. The improvements in the protocol make the activation process less frenetic, easier to control, more reproducible, and more amenable to scaling up.
This article describes the improved protocol for carrying out controlled CDAP activation of polysaccharide at 0 &#176;C and at a specified target pH and performing subsequent derivatization of the activated polysaccharides with ADH. Also described is a trinitrobenzene sulfonic acid (TNBS) assay, based on the method of Qi et al.9, for the determination of hydrazide level on the modified polysaccharide. A modified assay for hexoses based on resorcinol and sulfuric acid10 is also described, which can be used for determining a broader range of polysaccharides. For more information on CDAP activation and conjugation, the reader is referred to earlier publications5,6,8 by Lees et al.

<table><tbody><tr><td>Acetonitrile</td><td>Sigma</td><td>34851</td><td></td></tr><tr><td>Adipic acid dihydrazide</td><td>Sigma</td><td>A0638</td><td>MW 174</td></tr><tr><td>Amicon Ultra 15 10 kDa</td><td>Millipore</td><td>UFC901008</td><td>MW cutoff can be 30 kDa for 200 kDa PS</td></tr><tr><td>Analytical balance</td><td></td><td></td><td></td></tr><tr><td>Autotitrator or electronic pipet</td><td></td><td></td><td></td></tr><tr><td>Beaker&amp;nbsp; 2-4 L</td><td></td><td></td><td></td></tr><tr><td>CDAP</td><td>SAFC</td><td>RES1458C</td><td>Sigma</td></tr><tr><td>DMAP</td><td>Sigma</td><td>107700</td><td>MW 122.2</td></tr><tr><td>Flake ice</td><td></td><td></td><td></td></tr><tr><td>HCl 1 M</td><td>VWR</td><td>BDH7202-1</td><td></td></tr><tr><td>Micro stir bar</td><td>VWR</td><td>76001-878</td><td></td></tr><tr><td>Microfuge tube (for CDAP)</td><td>VWR</td><td>87003-294</td><td></td></tr><tr><td>NaCl</td><td>VWR</td><td>BDH9286</td><td></td></tr><tr><td>NaOH 1 M</td><td>Sigma</td><td>1099130001</td><td></td></tr><tr><td>NaOH 10 M</td><td>Sigma</td><td>SX0607N-6</td><td></td></tr><tr><td>pH meter</td><td></td><td></td><td></td></tr><tr><td>pH probe</td><td>Cole Parmer</td><td>55510-22</td><td>6 mm x 110 mm Epoxy single junction</td></tr><tr><td>pH temperature probe</td><td></td><td></td><td></td></tr><tr><td>Pipets &amp;amp; tips</td><td></td><td></td><td></td></tr><tr><td>Saline or PBS</td><td></td><td></td><td></td></tr><tr><td>Small beaker 5-20 mL</td><td>VWR</td><td>10754-696</td><td>A 10 mL beaker allows room for pH probe &amp;amp; pipet</td></tr><tr><td>Small ice bucket</td><td></td><td></td><td></td></tr><tr><td>Small spatula</td><td></td><td></td><td></td></tr><tr><td>Stir plate</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Resorcinol assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Combitip</td><td>Eppendorf</td><td>10 ml</td><td></td></tr><tr><td>DI water</td><td></td><td></td><td></td></tr><tr><td>Dialysis tubing</td><td>Repligen</td><td>132650T</td><td>Spectra/Por 6-8kDa</td></tr><tr><td>Dialysis tubing clips</td><td>Repligen</td><td>142150</td><td></td></tr><tr><td>Heating block</td><td></td><td></td><td></td></tr><tr><td>Nitrile gloves</td><td>VWR</td><td></td><td></td></tr><tr><td>Repeat pipettor</td><td>Eppendorf</td><td>M4</td><td></td></tr><tr><td>Resorcinol</td><td>Sigma</td><td>398047</td><td></td></tr><tr><td>Sugar standard</td><td></td><td>As appropriate</td><td></td></tr><tr><td>&amp;nbsp;Sulfuric acid 75%</td><td>VWR</td><td>BT126355-1L</td><td></td></tr><tr><td>Timer</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;TNBS assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adipic dihydrazide</td><td>Sigma</td><td>A0638</td><td>MW 174</td></tr><tr><td>Borosilcate test tubes 12 x 75</td><td>VWR</td><td>47729-570</td><td></td></tr><tr><td>Sodium borate, 0.5 M pH 9</td><td>Boston Biologicals</td><td>BB-160</td><td></td></tr><tr><td>TNBS 5% w/v</td><td>Sigma</td><td>P2297</td><td>MW 293.17</td></tr></tbody></table>

activation and conjugation of soluble polysaccharides using 1-cyano-4-dimethylaminopyridine tetrafluoroborate (cdap)

Conjugate vaccines are remarkable advances in vaccinology. For the preparation of polysaccharide conjugate vaccines, the polysaccharides can be conveniently functionalized and linked to vaccine carrier proteins using 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP), an easy-to-handle cyanylating reagent. CDAP activates polysaccharides by reacting with carbohydrate hydroxyl groups at pH 7-9. The stability and reactivity of CDAP are highly pH-dependent. The pH of the reaction also decreases during activation due to the hydrolysis of CDAP, which makes good pH control the key to reproducible activation. The original CDAP activation protocol was performed at room temperature in unbuffered pH 9 solutions.
Due to the rapid reaction under this condition (&#60;3 min) and the accompanying fast pH drop from the rapid CDAP hydrolysis, it was challenging to quickly adjust and maintain the target reaction pH in the short time frame. The improved protocol described here is performed at 0 &#176;C, which slows CDAP hydrolysis and extends the activation time from 3 min to ~15 min. Dimethylaminopyridine (DMAP) was also used as a buffer to pre-adjust the polysaccharide solution to the target activation pH before adding the CDAP reagent. The longer reaction time, coupled with the slower CDAP hydrolysis and the use of DMAP buffer, makes it easier to maintain the activation pH for the entire duration of the activation process. The improved protocol makes the activation process less frenetic, more reproducible, and more amenable to scaling up.

To illustrate the activation and derivatization of a polysaccharide using CDAP chemistry, dextran was activated at 0.25 and 0.5 mg CDAP/mg dextran. For each reaction, a 10 mg/mL dextran solution in water was chilled on ice, and 1/10th volume of a 2.5 M DMAP stock (prepared as described in section 3) was added. The final solution was brought to pH 9 by the addition of 0.1 M NaOH in 10 &#181;L aliquots. The solution was chilled and stirred, CDAP added, and the pH maintained at pH 9 by adding 10 &#181;L aliquots of 0.1 M NaOH for 15 min. Only 0.25 mL of 0.5 M ADH at pH 9 was added (less than the usual amount) and the reaction allowed to proceed overnight at 4 &#176;C.
The labeled dextran was then sequentially dialyzed against 1 M NaCl, 0.15 M NaCl, and water as described in section 6. The ADH-dextran was then assayed for dextran using the resorcinol/sulfuric acid assay (section 7.2). A typical standard curve using glucose as the sugar standard is shown in Figure 4A. The hydrazide content was determined using the TNBS assay described in section 7.3. A typical hydrazide standard curve using ADH as the standard is given in Figure 4B.
Representative calculations from the activation of dextran at the two levels of activation are shown in Figure 4A,B. The data are presented as both hydrazides per 100 kDa of dextran polymer and as a weight percent of ADH to dextran, as described in sections 7.9.3.4 and 7.9.3.5, respectively, in Figure 4C. The degree of derivatization approximately doubled as the CDAP ratio was doubled.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62597/62597fig01.jpg" /> 
Figure 1: Chemical structure of CDAP. CDAP = 1-cyano-4-dimethylaminopyridine tetrafluoroborate. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62597/62597fig02.jpg" /> 
Figure 2: Process of CDAP activation and conjugation. The process is conceptually divided into two phases, with the activated polysaccharide common to both. Under basic conditions, CDAP activates polysaccharide hydroxyls, releasing DMAP (reaction 1). CDAP hydrolysis also releases DMAP (reaction 3). Although a cyano-ester is shown, this may not be the actual intermediate. The intermediate is, therefore, referred to as (CDAP) &#34;activated&#34; polysaccharide. During the first activation phase, the activated polysaccharide can hydrolyze (reaction 4) or undergo side reactions (reaction 5). In the second conjugation phase (reaction 2), the activated polysaccharide reacts with an amine to form a stable isourea bond in addition to reactions 4 and 5. Abbreviations: CDAP = 1-cyano-4-dimethylaminopyridine tetrafluoroborate; DMAP = 4-dimethylaminopyridine; R-NH2 = amine. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62597/62597fig03.jpg" /> 
Figure 3: CDAP activation and conjugation. The process requires balancing CDAP reactivity with the polysaccharide, the stability of the CDAP and activated polysaccharide, as well as the reactivity of the activated polysaccharide with that of the amine. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62597/62597fig4v4.jpg" /> 
Figure 4: Representative results for CDAP activation of dextran. Typical standard curves for the (A) resorcinol/sulfuric acid and (B) TNBS assays. The assay results for dextran activated with 0.25 and 0.5 mg CDAP/mg dextran are shown. Glucose was used as the standard for the resorcinol assay. Dextran, in mg/mL, is divided by 100 kDa to give a molar concentration. The hydrazide concentration is determined using ADH as the standard and the results expressed as &#181;M Hz.&#160;(C)&#160;Calculation of hydrazide: dextran ratios.The level of derivatization was calculated as hydrazides per 100 kDa of dextran to facilitate the comparison between polymers of different average molecular weights. The % weight ratio of g ADH/g dextran was calculated using a MW of 174 g/mole for ADH. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig4v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate CDAP at JoVE.com

Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate CDAP

Proteins and amine-containing ligands can be covalently linked to polysaccharides activated by the cyanylation reagent, 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP), to form covalent protein (ligand)-polysaccharide conjugates. This article describes an improved protocol for carrying out controlled CDAP activation at 0 &#176;C and varying pH and performing subsequent conjugation of the activated polysaccharides.

Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP)

Conjugate vaccines are remarkable advances in vaccinology. For the preparation of polysaccharide conjugate vaccines, the polysaccharides can be ...

activation-conjugation-soluble-polysaccharides-using-1-cyano-4

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Immunology and Infection

6.3K Views.  Fina Biosolutions LLC. Proteins and amine-containing ligands can be covalently linked to polysaccharides activated by the cyanylation reagent, 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP), to form covalent protein (ligand)-polysaccharide conjugates. This article describes an improved protocol for carrying out controlled CDAP activation at 0 °C and varying pH and performing subsequent conjugation of the activated polysaccharides. 

Video: Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate CDAP

Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other fields. For instance, trehalose analogues bearing detectable tags have been used to detect Mycobacterium tuberculosis and may have applications as tuberculosis diagnostic imaging agents. Hydrolytically stable versions of trehalose are also being pursued due to their potential for use as non-caloric sweeteners and bioprotective agents. Despite the appeal of this class of compounds for various applications, their potential remains unfulfilled due to the lack of a robust route for their production. Here, we report a detailed protocol for the rapid and efficient one-step biocatalytic synthesis of trehalose analogues that bypasses the problems associated with chemical synthesis. By utilizing the thermostable trehalose synthase (TreT) enzyme from Thermoproteus tenax, trehalose analogues can be generated in a single step from glucose analogues and uridine diphosphate glucose in high yield (up to quantitative conversion) in 15-60 min. A simple and rapid non-chromatographic purification protocol, which consists of spin dialysis and ion exchange, can deliver many trehalose analogues of known concentration in aqueous solution in as little as 45 min. In cases where unreacted glucose analogue still remains, chromatographic purification of the trehalose analogue product can be performed. Overall, this method provides a &#34;green&#34; biocatalytic platform for the expedited synthesis and purification of trehalose analogues that is efficient and accessible to non-chemists. To exemplify the applicability of this method, we describe a protocol for the synthesis, all-aqueous purification, and administration of a trehalose-based click chemistry probe to mycobacteria, all of which took less than 1 hour and enabled fluorescence detection of mycobacteria. In the future, we envision that, among other applications, this protocol may be applied to the rapid synthesis of trehalose-based probes for tuberculosis diagnostics. For instance, short-lived radionuclide-modified trehalose analogues (e.g., 18F-modified trehalose) could be used for advanced clinical imaging modalities such as positron emission tomography-computed tomography (PET-CT).

Trehalose analogues are emerging as important molecules for bio(techno)logical and biomedical applications. We describe an optimized protocol for enzymatically synthesizing and purifying trehalose analogues that is simple, efficient, fast, and environmentally friendly. Its application to the rapid production and administration of a probe for the detection of mycobacteria is demonstrated.

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other ...

Biochemistry

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and efficient synthesis of well-controlled fluorescent statistical glycopolymers using reversible addition-fragmentation chain-transfer (RAFT)-based polymerization is demonstrated. The synthesis starts with the preparation of &#946;-galactose-containing glycomonomer 2-lactobionamidoethyl methacrylamide obtained by reaction of lactobionolactone and N-(2-aminoethyl) methacrylamide (AEMA). 2-Gluconamidoethyl methacrylamide (GAEMA) is used as a structural analog lacking a terminal &#946;-galactoside. The following RAFT-mediated copolymerization reaction involves three different monomers: N-(2-hydroxyethyl) acrylamide as spacer, AEMA as target for further fluorescence labeling, and the glycomonomers. Tolerant of aqueous systems, the RAFT agent used in the reaction is (4-cyanopentanoic acid)-4-dithiobenzoate. Low dispersities (&#8804;1.32), predictable copolymer compositions, and high reproducibility of the polymerizations were observed among the products. Fluorescent polymers are obtained by modifying the glycopolymers with carboxyfluorescein succinimidyl ester targeting the primary amine functional groups on AEMA. Lectin-binding specificities of the resulting glycopolymers are verified by testing with corresponding agarose beads coated with specific glycoepitope recognizing lectins. Because of the ease of the synthesis, the tight control of the product compositions and the good reproducibility of the reaction, this protocol can be translated towards preparation of other RAFT-based glycopolymers with specific structures and compositions, as desired.

An efficient, three-step synthesis of RAFT-based fluorescent glycopolymers, consisting of glycomonomer preparation, copolymerization, and post-modification, is demonstrated. This protocol can be used to prepare RAFT-based statistical glycopolymers with desired structures.

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and ...

Chemistry

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Bioengineering

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

One-pot Microwave-assisted Conversion of Anomeric Nitrate-esters to Trichloroacetimidates

The goal of the following procedure is to provide a demonstration of the one-pot conversion of a 2-azido-1-nitrate-ester to a trichloroacetimidate glycosyl donor. Following azido-nitration of a glycal, the product 2-azido-1-nitrate ester can be hydrolyzed under microwave-assisted irradiation. This transformation is usually achieved using strongly nucleophilic reagents and extended reaction times. Microwave irradiation induces hydrolysis, in the absence of reagents, with short reaction times. Following denitration, the intermediate anomeric alcohol is converted, in the same pot, to the corresponding 2-azido-1-trichloroacetimidate.

A 2-azido-1-nitrate-ester can be converted to the corresponding 2-azido-1-trichloroacetimidate in a one-pot procedure. The goal of the manuscript is to demonstrate utility of the microwave reactor in carbohydrate synthesis.

The goal of the following procedure is to provide a demonstration of the one-pot conversion of a 2-azido-1-nitrate-ester to a trichloroacetimidate ...

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having multifarious bioactivities. Although chemical O-glycosylation is a commonly beneficial strategy to synthesize disaccharide nucleosides, the preparation of substrates such as glycosyl donors and acceptors requires&#160;tedious protecting&#160;group manipulations and a purification at each synthetic step. Meanwhile, several research groups have reported that boronic and borinic esters serve as a protecting or activating group of carbohydrate derivatives to achieve the regio- and/or stereoselective acylation, alkylation, silylation, and glycosylation. In this article, we demonstrate the procedure for the regioselective O-glycosylation of unprotected ribonucleosides utilizing boronic acid. The esterification of 2&#39;,3&#39;-diol of ribonucleosides with boronic acid makes the temporary protection of diol, and, following O-glycosylation with a glycosyl donor in the presence of p-toluenesulfenyl chloride and silver triflate, permits the regioselective reaction of the 5&#39;-hydroxyl group to afford the disaccharide nucleosides. This method could be applied to various nucleosides, such as guanosine, adenosine, cytidine, uridine, 5-metyluridine, and 5-fluorouridine. This article and the accompanying video represent&#160;useful (visual) information for the O-glycosylation of unprotected nucleosides and their analogs for the synthesis of not only disaccharide nucleosides, but also a variety of biologically relevant derivatives.

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Here, we present protocols for the synthesis of disaccharide nucleosides by the regioselective O-glycosylation of ribonucleosides via a temporary protection of their 2&#39;,3&#39;-diol moieties utilizing a cyclic boronic ester. This method applies to several unprotected nucleosides such as adenosine, guanosine, cytidine, uridine, 5-methyluridine, and 5-fluorouridine to give corresponding disaccharide nucleosides.

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having ...

Hierarchical and Programmable One-Pot Oligosaccharide Synthesis

This article presents a general experimental protocol for programmable one-pot oligosaccharide synthesis and demonstrates how to use Auto-CHO software for generating potential synthetic solutions. The programmable one-pot oligosaccharide synthesis approach is designed to empower fast oligosaccharide synthesis of large amounts using thioglycoside building blocks (BBLs) with the appropriate sequential order of relative reactivity values (RRVs). Auto-CHO is a cross-platform software with a graphical user interface that provides possible synthetic solutions for programmable one-pot oligosaccharide synthesis by searching a BBL library (containing about 150 validated and &#62;50,000 virtual BBLs) with accurately predicted RRVs by support vector regression. The algorithm for hierarchical one-pot synthesis has been implemented in Auto-CHO and uses fragments generated by one-pot reactions as new BBLs. In addition, Auto-CHO allows users to give feedback for virtual BBLs to keep valuable ones for further use. One-pot synthesis of stage-specific embryonic antigen 4 (SSEA-4), which is a pluripotent human embryonic stem cell marker, is demonstrated in this work.

This protocol demonstrates how to use the Auto-CHO software for hierarchical and programmable one-pot synthesis of oligosaccharides. It also describes the general procedure for RRV determination&#160;experiments and one-pot glycosylation of SSEA-4.

This article presents a general experimental protocol for programmable one-pot oligosaccharide synthesis and demonstrates how to use Auto-CHO software for ...

Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry

The recent rise in virus-like particles (VLPs) in biomedical and materials research can be attributed to their ease of biosynthesis, discrete size, genetic programmability, and biodegradability. While they&#39;re highly amenable to bioconjugation reactions for adding synthetic ligands onto their surface, the range in bioconjugation methodologies on these aqueous born capsids is relatively limited. To facilitate the direction of functional biomaterials research, non-traditional bioconjugation reactions must be considered. The reaction described in this protocol uses dibromomaleimides to introduce new functionality in the solvent exposed disulfide bonds of a VLP based upon Bacteriophage Q&#946;. Furthermore, the final product is fluorescent, which has the added benefit of generating a trackable in vitro probe using a commercially available filter set.

Here, we present a procedure to fluorescently functionalize the disulfides on Q&#946; VLP with dibromomaleimide. We describe Q&#946; expression and purification, the synthesis of dibromomaleimide-functionalized molecules, and the conjugation reaction between dibromomaleimide and Q&#946;. The resulting yellow fluorescent conjugated particle can be used as a fluorescence probe inside cells.

The recent rise in virus-like particles (VLPs) in biomedical and materials research can be attributed to their ease of biosynthesis, discrete size, ...

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic fever and autoimmune diseases. Since thousands of antibodies with different epitopes are circulating in blood, no universal method, except for the dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF), was reported to target specific harmful antibodies. The development of DCAF molecules makes significant contribution to the progress of targeted therapy, which were demonstrated to eliminate the antibody dependent enhancement (ADE) effect in a Dengue virus (DENV) infection model and to boost the acetylcholine receptor activity in a myasthenia gravis model. Here, we describe a protocol for the synthesis of a DCAF molecule (DCAF1), which can selectively block 4G2 antibody to attenuate ADE effect during Dengue virus infection, and illustrate the binding of DCAF1 to 4G2 antibody by an ELISA assay. In our method, DCAF1 is synthesized by the conjugation of a hydrazine derivative of a Fc-III peptide and a recombinant expressed long &#945;-helix with antigenic sequence through native chemical ligation (NCL). This protocol has been successfully applied to DCAF1 as well as other DCAF molecules for targeting their cognate antibodies.

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics DCAF

Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) is novel for the elimination of harmful antibodies. Here, we describe a detailed protocol for the synthesis of DCAF1 molecule, which can selectively block 4G2 antibody to eliminate antibody dependent enhancement effect during Dengue virus infection.

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP)

Summary

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Chapters in this video

Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

One-pot Microwave-assisted Conversion of Anomeric Nitrate-esters to Trichloroacetimidates

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Hierarchical and Programmable One-Pot Oligosaccharide Synthesis

Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis